Summary/Abstract: TGF-? drives the fibroinflammatory processes that leads to lung and airway fibrosis. The long-term goal of this project is to acquire a deep understanding of the regulation of TGF-? activity to develop new strategies and treatments for fibrosing lung disease. There are few effective therapies to treat chronic fibrosing and inflammatory diseases of the lung. The cytokine TGF-? is a central mediator of fibrosis and pathologic inflammation and is a potential therapeutic target in fibrosing lung disease. However, the practical utility of targeting TGF-? itself or its receptors is limited by risk of toxicities seen in rodents, primates and humans. More specific methods to target the fibroinflammatory effects of TGF-? are highly desirable. A promising method to more specifically target local effects of TGF-? is to target its ?activation? since it is always produced in a latent form (L-TGF-?) that must be activated in order to function. Another feature of L-TGF-? that could facilitate more specific targeting is that it is covalently bound to specific cell surfaces by GARP. L-TGF-? binding to the integrin ?v?8 is essential for TGF-? activation in vivo. For the ?v?8 activation mechanism, as well as all others, it has long been assumed that TGF-? must be released from LAP so that free TGF-? can diffuse and bind its receptors on target cells. Based on recent structural data obtained using single particle electron cryomicroscopy (cryo-EM), we have recently proposed a new model whereby ?v?8 can bind to L- TGF-? on cells presenting the L-TGF-?/GARP complex and induce signaling without release and diffusion of TGF-?. Here in three aims, we address three critical questions concerning this new model of L-TGF-? activation. (1) Which flexible domains of L-TGF-? of the ?v?8/L-TGF-?/GARP ternary complex shield TGF-? from its receptors? (2) Is flexibility of L-TGF-? induced by binding to ?v?8 necessary to mediate TGF-? activation? (3) Do TGF-? receptors (TGF-?Rs) bind to TGF-? within the ?v?8/L-TGF-?/GARP complex? To answer these questions, we will use cryo-EM to determine the structure of the ?v?8/L-TGF-?/GARP complex to determine how flexibility of L-TGF-? contributes to TGF-? activation, and finally we will capture the multimeric complex of TGF-?Rs with ?v?8/LTGF-?/GARP. These studies will improve mechanistic understanding of TGF-? activation and therapeutic targeting strategies to inhibit it.